Production of protein-polysaccharide conjugates

ABSTRACT

The present invention provides novel compositions and methods for producing protein-polysaccharide conjugates in aqueous solutions. Also provided are methods for limiting the Maillard reaction to the very initial stage, the formation of the Schiff base. Provided are methods to obtain a simple product of Schiff base with white color, and compositions obtained using the methods of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This invention claims priority to U.S. Provisional Patent ApplicationSer. No. 61/037,912, filed Mar. 19, 2008, which is herein incorporatedby reference.

GOVERNMENT INTERESTS

This invention was made with United States government support awarded bythe United States Department of Agriculture, grant No. 2004-35503-14839.The United States government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates generally to the field ofprotein-polysaccharide conjugates.

BACKGROUND

Food proteins and polysaccharides (PS) are two key structural componentsused to control texture, structure, and stability of food materials.Typically, foods contain both biopolymers in the form of complexmulticomponent mixtures. Conjugation of protein with polysaccharide hasbeen intensively studied in recent years (Dunlap and Côté, 2005, J.Agric. Food Chem. 53: 419-423), with a significant improvement infunctional properties (e.g. solubility and heat stability) over a widepH range.

Protein-polysaccharide conjugates (PPC) (i.e., proteins covalentlylinked to polysaccharides) are useful as emulsifiers in foods andbeverages. The addition of the polysaccharide stabilizes the protein,and such protein-polysaccharide conjugates have superior functionalproperties (e.g., gelation, emulsification, solubility, heat/pHstability) compared to the unaltered proteins. A naturally producedprotein-polysaccharide conjugate can be found in gum arabic, whichcontains about 2% covalently bound protein. Gum arabic is usedextensively as a natural food emulsifier/stabilizer for emulsions andbeverages, as an encapsulation agent for flavor delivery, in gum dropsand similar candies, and to control ice crystallization in frozenproducts. However, the price and availability of gum arabic is extremelyvolatile due to a variety of growth, harvest, and regional issues. Abouthalf of the gum arabic produced worldwide is imported by the US(approximately 30,000 tons). Consequently, there is considerablecommercial interest in developing a substitute for gum arabic.

Two main techniques are used to produce covalently linkedprotein-polysaccharide conjugates: (1) chemical modification usingreagents such as carbodiimide, and (2) glycation by exploiting theinitial step (i.e., Schiff base step which is well before the browningand other undesirable reactions) of the Maillard reaction between areducing sugar (e.g., glucose, lactose) and an amino group (e.g.,lysine). Chemical modifications usually use toxic reagents that are notdesirable for use in food ingredients. The usual glycation procedureinvolves using dry heating (e.g., 60° C.), and storage of thelyophilized mixtures (protein and PS) for a period of up to severalweeks at a specific relative humidity (RH).

The initial step in the Maillard reaction, the formation of a covalentlinkage between ε-NH₂ amino groups on proteins and carbonyl groups onreducing sugars, has been used to create new ingredients with improvedfood functionalities. The Maillard reaction is comprised of a complexseries of reactions, which simultaneously occur by multiple reactionpathways. Generally, the Maillard reaction occurs in three stages(Hodge, 1953, J. Agric. Food Chem. 1: 928-943). The initial stageconsists of the condensation between ε-NH₂ amino groups and carbonylgroups, Schiff base formation and irreversible Amadori rearrangement,which leads to the Amadori products. The products at this initial stageare colorless, and there is no absorption in the near-ultravioletspectrum. The intermediate stage involves sugar dehydration, sugarfragmentation and amino acid degradation. These products result inabsorption at 277-285 nm due to the furfural region (Vallejo-Cordoba andGonzalez-Cordova, 2007, Electrophoresis 28: 4063-4071; Hodge, 1953, J.Agric. Food Chem. 1: 928-943). Products are colorless or yellow in theintermediate stage. The final stage is highly colored (yellow or brown),with the formation of brown pigments called melanoidins.

The conjugation of proteins with polysaccharides (PS) is usually carriedout using dry-heat treatment, for example the conjugation ofβ-lactoglobulin (β-Ig) and dextran, under conditions of about 60° C.,35-40% RH (relative humidity), for about 3 weeks (Dickinson and Galazka,1991, Food Hydrocoll. 5: 281-296). However, there are severaldisadvantages for this method. It requires powdered protein and PSmaterials and a constant temperature and relative humidity (e.g. 79%RH), which must be maintained during the reaction. The time required forthe reaction is also significant (e.g. 3-5 weeks at 60° C.). Thereaction process also cannot be easily controlled and the products arecomplicated. Intermediate or final stage products of the Maillardreaction are also typically obtained as indicated by the appearance ofundesirable light yellow or brown color, which results in increasedabsorbance at ≥420 nm (Dickinson and Galazka, 1991; Tanaka et al., 1999,Fisheries Sci. 65: 623-628). Although the conjugation of proteins withPS by the dry heating process has resulted in interesting improvementsin functional properties, the dry-heat treatment method is notattractive and, as a result of the significant disadvantages describedabove, there are few commercial PPC ingredients.

Whey proteins, including β-Ig, have been used to stabilize foodemulsions because of their surface active properties (Dickinson, 1997,J. Dairy Sci. 80: 2607-2619). The emulsifying properties of PPC preparedwith whey proteins by the dry heating process have been studiedextensively. While many types of PS have been tested by this dry heatingprocess little is known about the effect of the molecular weight size ofthe PS on the properties of the PPC.

To use the Maillard conjugation type of approach to commercially produceviable food ingredients, new methods are needed to induce this reactionin a short processing time and in aqueous protein-PS mixtures instead ofthe expensive lyophilized samples that have been previously studied. Itwould be advantageous to formulate new ingredients (e.g., PPC) thatprovide improved functionality (e.g., emulsion stability) and healthbenefits in various nutritional products (e.g., reduced allergenicity ininfant formulae and reduced astringency in low pH protein-fortifiedbeverages). The present invention addresses these and related needs.

BRIEF SUMMARY

Methods of preparing polysaccharide-protein conjugates are provided. Themethods include: reacting polysaccharides comprising a reducing sugarand proteins in an aqueous solution comprising 10-40% (w/v)polysaccharide and 1-30% (w/v) protein, under temperature conditions offrom about 40° C. to about 120° C., thereby producingpolysaccharide-protein conjugates. In the practice of the methods, thesolutions may be acidified to a pH from about 6.0 to about 8.0. Thereducing sugar and the proteins may be reacted under temperatureconditions of from about 40° C. to about 120° C. for a period of fromabout 1 hour to about 48 hours. The solutions may be subjected to ahydrostatic pressure in the amount of between about 1 MPa and about 20MPa. The methods may further include recovering of thepolysaccharide-protein conjugates from the solutions in various ways,for example including chromatography. Detection of thepolysaccharide-protein conjugates may be performed by difference UVspectroscopy. The difference UV spectroscopy may include measuringabsorbance of the polysaccharide-protein conjugates at a wavelength of304 nm. In the practice of the methods, the proteins may be, forexample, soy proteins, or they may be caseinates. Methods ofemulsification, comprising using as emulsifiers the abovepolysaccharide-protein conjugates are also provided.

Methods for producing whey protein isolate (WPI)-dextran conjugates areprovided. The methods include: reacting dextran and whey protein isolatein an aqueous solution comprising 10-40% (w/v) dextran and 1-30% (w/v)protein, under temperature conditions of from about 40° C. to about 120°C., thereby producing whey protein isolate-dextran. In the practice ofthe methods, the solutions may be acidified to a pH from about 6.0 toabout 8.0. The whey protein isolate and the dextran may be reacted undertemperature conditions of from about 40° C. to about 120° C. for aperiod of from about 1 hour to about 48 hours. The solutions may besubjected to a hydrostatic pressure in the amount of 1-20 MPa. Themethods may include the steps of recovering the whey proteinisolate-dextran conjugates from the solutions, for example includingchromatography. Detection of the whey protein isolate-dextran conjugatesmay be performed by difference UV spectroscopy. The difference UVspectroscopy may include measuring absorbance of the whey proteinisolate-dextran conjugates at a wavelength of 304 nm. Whey proteinisolate-dextran conjugates which are obtained by these methods areprovided. Methods of emulsification, comprising using as emulsifiers theabove whey protein isolate-dextran conjugates are also provided.

Polysaccharide-protein conjugates are provided, where the conjugates arecomprised substantially of Schiff base. The conjugates may besubstantially free of intermediate and advanced Maillard products. Theconjugates may be white in color, and they may maintain the white colorfor a period of at least three months. The conjugates are able toproduce fat globules during homogenization that are of small particlesize, e.g. the fat globules may be less than about 2 μm in diameter. Theconjugates may be thermostable for a period of at least 3 weeks insolutions that are held at about 5° C. The stability of the emulsion canbe determined by the lack of a large change in particle size duringstorage of the emulsion.

Emulsifying compositions and methods of emulsification are provided. Theemulsifying compositions and the methods include using as emulsifyingcompositions the polysaccharide-protein conjugates of the presentinvention.

Food compositions with alleviated astringency and methods of alleviatingastringency of proteins are provided. The food compositions and themethods include using as astringency alleviating compositions thepolysaccharide-protein conjugates of the present invention.

Food compositions with reduced allergenicity and methods of producingfood compositions with reduced allergenicity are provided. The foodcompositions with reduced allergenicity and the methods include using asreduced allergenicity compositions the polysaccharide-protein conjugatesof the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows images of SDS-PAGE of WPI-dextran conjugates in the absence(lanes 1-4) and presence (lanes 5-8) of 5% of 2-mercaptoethanol. PanelA: protein stain (Coomassie blue). Panel B: carbohydrate stain (periodicacid). Mr: molecular weight standards. Lanes 1 and 5, 10% WPI(unheated). Lanes 2 and 6, mixture of 10% WPI-30% dextran (unheated, pH6.5, 60° C., 24 h). Lanes 3 and 7, 10% WPI (pH 6.5, 60° C., 24 h). Lanes4 and 8, mixture of 10% WPI-30% dextran (pH 6.5, 60° C., 24 h).

FIG. 2 is a graph showing the difference in UV absorbance spectra (DUV)of Schiff base of WPI-dextran conjugates. Curve 1: 30% dextran (60° C.for 24 h). Curve 2: 10% WPI (60° C. for 24 h). Curve 3: mixture of 10%WPI-30% dextran (60° C. for 24 h).

FIG. 3 is a graph showing the time course of WPI-dextran Schiff baseformation as indicated by the DUV peak at 304 nm (10% WPI-30% dextran,pH 6.5, 60° C. for 24 h, n=3).

FIG. 4 is a graph showing the effect of WPI concentration on theWPI-dextran Schiff base formation (30% dextran, pH 6.5, 60° C. for 24 h,n=3).

FIG. 5 is a graph showing the effect of dextran concentration on theWPI-dextran Schiff base formation (10% WPI, pH 6.5, 60° C. for 24 h,n=3).

FIG. 6 is a graph showing the effect of temperature [40° C. (∘); 50° C.(□); 56° C. (Δ); 60° C. (⋄)] and pH on the WPI-dextran Schiff baseformation (10% WPI-30% dextran, 60° C. for 24 h, n=3).

FIG. 7 is a graph showing the effect of hydrostatic pressure on theWPI-dextran Schiff base formation at various pH values (10% WPI-30%dextran, 60° C. for 24 h, n=3).

FIG. 8 is a graph illustrating the emulsifying properties ofprotein-polysaccharide conjugates produced according to the presentinvention.

FIG. 9 is a graph showing the solubility of conjugates (0.1% protein) atroom temperature and heated 30 min at 80° C. within the pH range3.5-7.5, in comparison with whey protein (0.1% WPI). The higher theabsorbance at 500 nm, the lower the solubility is.

FIG. 10 is a graph showing the solubility of conjugates (0.1% protein,pH 4.5) at room temperature and heated 30 min at 80° C., in comparisonwith whey protein (0.1% WPI). The higher the absorbance at 500 nm, thelower the solubility is.

FIG. 11 is a graph showing thermal stability of the conjugate, as a DSCheating scan (1.0° C./min) for 10% conjugate (dotted line) and 10% WPI(solid line) at pH 8.5.

FIG. 12 is a graph showing the effect of solids content of a mixture ofsodium caseinate: maltodextrin (1:1 ratio) on the formation of Schiffbase, as indicated by the ΔA305 nm or change in difference UVspectroscopy (DUV) at 305 nm and color (ΔA450 nm).

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

In one aspect, the present invention relates to protein-polysaccharideconjugates (PPC) and to methods of preparing same. In one example, thepresent invention provides novel methods to produceprotein-polysaccharide conjugates using an aqueous solution and using awet heat treatment. The invention is particularly well-suited forproducing protein-polysaccharide conjugates that may be used asemulsifiers.

“Aqueous solution” means a solution in which the solvent is primarilywater.

“Protein” means organic compounds made of amino acids arranged in alinear polymeric chain and joined together by peptide bonds between thecarboxyl and amino groups of adjacent amino acid residues.

“Polysaccharides” means complex carbohydrate polymers comprisingmultiple monosaccharide units joined together by glycosidic bonds.Polysaccharides have a general formula of Cn(H₂O)_(n-1) where n isusually a large number between 200 and 2500.

“Protein-polysaccharide conjugates” (PPC) refers to proteins covalentlylinked to polysaccharides.

“Reducing sugar” means any sugar that, in basic solution, forms analdehyde or ketone. This allows the sugar to act as a reducing agent,for example in the Maillard reaction. Reducing sugars include, but arenot limited to, glucose, fructose, glyceraldehyde, lactose, arabinoseand maltose, maltodextrin, dextran.

“Dextran” means a complex, branched polysaccharide comprising multipleglucose molecules joined into chains of varying lengths (from 10 to 150kDa). The straight chain consists of α1→6 glycosidic linkages betweenglucose molecules, while branches begin from aα1→4 linkages (and in somecases, α1→2 and α1→3 linkages as well).

“Dextrin” means a low-molecular-weight carbohydrate produced by thehydrolysis of starch. Dextrins are mixtures of linear α-(1,4)-linkedD-glucose polymers starting with an α-(1,6) bond. An example of dextrinsis maltodextrin, a polysaccharide that is used as a common foodadditive.

“Schiff base” (or azomethine), named after Hugo Schiff, means afunctional group that contains a carbon-nitrogen double bond with thenitrogen atom connected to an aryl or alkyl group—but not hydrogen.Schiff bases are of the general formula R₁R₂C═N—R₃, where R₃ is an arylor alkyl group that makes the Schiff base a stable imine. In one examplethe methods of the present application may be used to control the extentof the Maillard reaction to stop at the initial Schiff base formation.Identification and/or quantification of Schiff base can be performedspectroscopically and/or calorimetrically.

Methods to produce protein-polysaccharide complexes using wet heattreatment are provided. In contrast to previous work on PPC made by thedry heating process, the present invention contemplates the use ofaqueous solutions to prepare PPC. These aqueous solutions may be aqueousmixtures. In one example the methods involve heating aqueous solutionsof relatively high concentrations of protein (e.g., 1-30%, preferably5-20%, and more preferably about 10% w/v protein) in the presence ofrelatively high concentrations of a polysaccharide with a reducing sugar(e.g., 10-40%, preferably 20-35%, and more preferably about 30% w/vpolysaccharide). The polysaccharide may include one type ofmonosaccharide unit (it may be a homopolysaccharide). Alternatively, thepolysaccharide may include more than one type of monosaccharide unit (itmay be a heteropolysaccharide).

A variety of proteins may be used for practicing the present invention.Useful proteins will preferably have one or more lysine residues. In oneexample, there is a minimum of one lysine reside per protein molecule.One lysine molecule may be sufficient to bind to one polysaccharidegroup, thereby creating a conjugate. In some examples, the proteins usedin the practice of the present invention have relatively small molecularweight. Non-limiting examples of proteins useful for practicing thepresent invention include a variety of proteins with molecular weightsin the range of about 11,000 Da to about 500,000 Da. The proteins mayinclude whey proteins (e.g. whey protein isolate, WPI), caseins(caseinates), soy proteins, etc. The amounts of WPI may vary, and insome examples the concentration of WPI that is used for practicing thepresent invention is typically in the range of about 1-30% (w/v), andpreferably the amounts of WPI are in the range of 5-20% (w/v). In oneexample, conjugates are formed in reactions that use starting aqueoussolutions of about 10% (w/v) WPI. As well, a variety of sugars(saccharides), preferably with one or more types of reducing terminal,may be used for practicing the present invention. Examples of sugarsthat can be used for practicing the present invention include:monosaccharides (e.g., glyceraldehyde; arabinose; ribose; xylose;galactose, glucose, mannose; fructose); disaccharides (e.g., lactose;maltose); polysaccharides (e.g., galactomannans; dextran; maltodextrin;chitosan; alginic acid; agar; carrageenan; dextran sulfate; konjacmannan; xyloglucan; starch; modified starches; pectins; dietary fiber,such as polydextrose, wheat dextrin, oat bran concentrate). In someexamples, the polysaccharides useful for practicing the presentinvention may include aqueous solutions of about 20-40% (w/v) dextran orabout 20-40% (w/v) maltodextrins, preferably about 30% (w/v) dextran orabout 30% (w/v) maltodextrin. The choice of polysaccharide influencesthe concentration of polysaccharide (w/v) used in the conjugationreaction. The concentration of polysaccharide influences the viscosityof the aqueous polysaccharide solution.

In some examples, to provide the mixture where the conjugation reactiontakes place, protein and polysaccharide may be provided as aqueoussolutions. Solvent may also be provided as necessary, to adjust theconcentrations of each of the protein and the polysaccharide to desiredvalues (w/v). Aqueous solutions of protein and of polysaccharide may beadded simultaneously, or they may be added sequentially. Once proteinand polysaccharide have been combined to create a mixture, the pH of themixture solution may be adjusted to a desired value using acids and/orbases.

Protein denaturation is a function of the temperature and the pH of theaqueous solution where the protein is located. While higher temperaturemay generally accelerate the conjugation reaction, higher temperaturealso may cause heat denaturation of the proteins. For some non-globularproteins like caseins there is no real denaturation temperature andhigher processing temperatures can be used compared with globularproteins. For example, the conjugation reaction is promoted by raisingthe temperature from 20° C. to 70° C. Not wanting to be bound by thefollowing theory, it is believed that the elevated concentration ofpolysaccharide may protect the protein from heat denaturation. Thepolysaccharides may act not only as a reactant taking part in theconjugation, but also as a protective reagent in preventing excessiveprotein denaturation and/or aggregation. Not wanting to be bound by thefollowing theory, it is believed that high polysaccharide concentrationmay also “crowd” the protein promoting the conjugation reaction. In somepreferred embodiments, polysaccharide such as dextran may be used as acrowding agent, while another type of reducing sugar may be used todrive the reaction. Alternatively, or in addition to dextran, othercrowding agents may be used instead of polysaccharides. Examples ofcrowding agent useful in the practice of the present invention include,but are not limited to, ficolls, dextrans, polyethylene glycol,polyvinyl alcohol, bovine serum albumin, and poly(vinylpyrrolidone).

The conjugation reactions may be carried out under controlledtemperature conditions. For example, the conjugation reaction may becarried out using temperatures in the range of about 40° C. to about120° C., and more preferably in the range of about 50° C. to about 70°C. In one example, the conjugation reaction may be carried out using atemperature of about 65° C. Higher temperatures are used for proteinsthat have higher denaturation temperatures than the WPI used in thisexample.

In one example, the conjugation reaction of the present invention may beinfluenced by controlling the pH of the reaction solution. Higher (morealkaline) pH values can shorten the time of the conjugation reaction.For example, for WPI the conjugation reaction is promoted by loweringthe pH from 8.5 to 5.5. It is believed that high pH values promotedisulfide protein interactions in whey proteins, which is undesirablefor the conjugation reaction.

In general, the specific temperatures and pH values for the conjugationreaction will depend on the particular protein used. The duration of thereaction may also depend on the particular protein used. For example,caseins are not very sensitive to heat denaturation so it is possible touse relatively high temperatures and relatively high pH as well, andthus obtain PPC in a shorter time. In such examples, when caseinate isthe reacted protein, higher temperatures and different pH conditions canbe used, e.g. temperatures for the conjugation reaction can be in therange of from about 40° C. to about 110° C., the reaction can beconducted for between 30 min to 4 hours, and the pH values can varybetween about 6.0 to about 8.5.

Hydrostatic pressure treatment may be used to control the conjugationreaction. For example, protein-polysaccharide mixtures may bepressurized at 2-20 MPa, preferably at about 7.9 MPa, by subjecting themixtures to pressure provided by cylinders of compressed nitrogen gas.

Once the proteins and the polysaccharides are mixed together, theconjugation reactions may be carried out for various periods of time.The duration of the reaction influences the yield of product, such asthe conjugate. It is contemplated that generally the conjugationreactions of the present invention are carried out for a period of timeof between 1 and 96 hours, and more preferably the conjugation reactionsof the present invention are carried out for a period of time of about 6to about 24 hours.

The conjugates produced according to the present invention may be useddirectly, without purification. Thus, the entire mixture may be used,for example as a food ingredient.

The methods of the present invention can be used to attach biologicallyimportant carbohydrates to proteins for desired nutritional and/orbioactivity purposes.

Optionally, the conjugates of the present invention may be purified. Theresulting conjugates may be purified using methods known in the art, forexample using chromatographic methods. Thus, PPC may be purified usinganion exchange chromatography and size exclusion chromatography (Dunlapand Côté, 2005, J. Agric. Food Chem. 53: 419-423) and/or affinitychromatography. In one example, the method of the present inventionprovide for obtaining substantial quantities of purified conjugates foruse in foods. The present invention also provides spectroscopic methodsfor monitoring the progress of the conjugation reactions andchromatographic methods for purifying the conjugates (conjugatecomplexes). In practice, purification of conjugates is not required ifthe reaction mixture is used as an ingredient.

To assist in the process for conjugating a protein and a polysaccharide,a crosslinker or a spacer can be provided on either the protein or thepolysaccharide. Because the crosslinker is a smaller molecule, it helpsthe coupling reaction for the larger protein and polysaccharidemolecules proceed more quickly by allowing better access to the largemolecules, and thereby enhancing the reactivity. Additionally, the useof a crosslinker allows one to more effectively control the degree ofcrosslinking and the chemical structure of the resultant conjugate.

Various procedures and chemistries are available for activating andattaching spacers to proteins and to polysaccharides, e.g., using CDAP,carbodiimides, NHS esters, CNBr, and carbodiimide. Published PCT PatentApplication No. WO/1997/041897, incorporated herein by reference,describes the use of vinylsulfones as the reactive group in acrosslinking agent.

The methods of the present invention may be practiced using aqueoussolutions and mixtures in a variety of volumes. For example, conjugationreactions may be performed in various vessels, beakers, tanks, etc. Themethods of the present invention may be practiced using batchprocessing.

The resulting purified conjugates (PPC) exhibit improved thermalstability, more desirable color (white as opposed to yellow or brown),and excellent emulsifying properties that are superior to both theunmodified whey protein and the gum arabic. White color means having thecolor of pure snow or milk, the color of radiated, transmitted, orreflected light containing all of the visible rays of the spectrum;opposite to black. The white color of the PPC conjugates of the presentinvention is stable and is maintained for months. In contrast,comparable compositions of conjugates that include other sugars turndarker within days of synthesis, exhibiting non-white, darker, yellow orbrown color.

The conjugates according to the present invention are able to producefat globules during homogenization that are of a small particle size,e.g. less than about 2 μm in diameter. The conjugates may bethermostable for a period of at least 3 weeks in solutions held at about5° C. The stability of the emulsion can be determined by the lack of alarge change in particle size during storage of the emulsion. Forexample, the denaturation temperature of the conjugates is about 87° C.by the determination using Differential Scanning Calorimetry (DSC),which is much higher than the denaturation temperature of Whey ProteinIsolate, which is about 72° C.

The compositions produced according to the present invention, such asunfractionated reaction mixtures containing PPC or the purified PPC, mayfunction as superemulsifiers, i.e. emulsifiers with properties superiorto gum arabic. Not wanting to be bound by the following theory, it ispossible that the steric stability is conferred by the bulky PS whilethe attached protein is able to give the hydrophobic/hydrophiliccharacter necessary to stabilize the emulsion interface. The conjugatesmay significantly decrease the interfacial tension, and improve thesolubility at severe heat treatments, lower pH, and higher saltconcentration than whey protein. One possible use of these emulsifiersis as fat replacers in low-fat foods. The conjugates may possess thefollowing emulsifying properties: a whey protein-dextran conjugate, madefrom dextran (molecular weight 500,000 kDa), is capable of producingfine emulsion droplets (0.25 μm diameter) with soybean oil, whereas theequivalent emulsion made with gum arabic produces droplets of 0.56 μmand WPI produces droplets of 0.31 μm. The emulsion stabilized byconjugates of the present invention is stable over a storage period ofat least up to 8 weeks, with no visible precipitation or phaseseparation, but emulsion made using whey protein or Gum Arabic is notstable over such period of time. Examples of methods for measuringemulsifying properties and determining the stability of emulsions arefound below.

The compositions produced according to the present invention mayalleviate astringency of proteins. “Astringency” refers to a complexgroup of sensations involving dryness, roughness of oral surfaces andtightening, drawing or puckering of the mucosa and muscles around themouth. Astringency has been attributed primarily to interactions betweenthe salivary proline-rich-proteins (SPRP) and other compounds that havea particularly high affinity for the SPRPs, such as polyphenols.Attachment of dextran onto a protein molecule could interfere with thereaction between proteins with salivary proteins and theirprecipitation. This could reduce the sensation of protein astringency inlow pH environments (e.g. as in protein fortified acid beverages).Conjugation of proteins alters their solubility (increases it) and alsotheir charge both of which could influence possible reactions withsalivary proteins. For example, glycosylated salivary proteins-tannincomplexes lead to a lower astringency perception (Sarni-Machado et al.,2008, J. Agric. Food Chem. 56: 9563-9569). Astringency of thecompositions of the present invention can be measured, e.g., using themethods described in Beecher et al., 2008, J. Dairy Sci. 91: 2553-2560.Examples of methods for determining astringency of proteins are foundbelow.

The compositions produced according to the present invention may beuseful in processing proteins such as whey protein isolate to make itless allergenic. Food allergy encompasses a group of disorderscharacterized by immunologic responses to food proteins. For example,conjugation of milk proteins with dextran may reduce their allergenicityby interfering with recognition sites on proteins that cause elevationof IgE. The reduced allergenicity of WPI may be of interest, forexample, to infant formula manufacturers. Examples of methods fordetermining reduced allergenicity are found below.

The term “reduced allergenicity” indicates that the amount of producedIgE (in humans, and molecules with comparable effects in specificanimals), which can lead to an allergic state, is decreased whenconsuming the protein-polysaccharide conjugates of the invention incomparison to comparable protein-polysaccharide conjugates obtainedaccording to other methods known in the art.

The compositions produced according to the present invention may beuseful in the incorporation of “biologically functional”carbohydrate/polysaccharide groups, e.g. groups that may confer thesensation of satiety (fullness after consumption, reducing appetite).

The compositions produced according to the present invention provide lowcost and plentiful emulsifying agents for food products (e.g., saladdressings, gum, gummy candies, frozen foods, etc.) and beverages (e.g.,Coke); substitute for gum arabic. Examples of whey protein emulsifiersand emulsions are described, for example, in published PCT PatentApplication No. WO/2006/090110 A1, which is incorporated herein byreference.

The compositions produced according to the present invention may be usedas vaccine adjuvants, using for example the approaches described inPetrovsky et al., Aug. 1, 2007, Biopharm, pp. 24-33; and in U.S. Pat.No. 6,573,245.

In one example, WPI-dextran conjugates can be formed in mixtures of 10%WPI-30% dextran during incubation at pH 6.5 and 60° C. for 24 h. Theconjugation of WPI and dextran can be confirmed using a variety ofmethods known in the art, including, for example, by SDS-PAGE with bothprotein staining and carbohydrate staining. The WPI-dextran conjugatecan be further identified to be mainly composed of Schiff base, which ischaracterized by a maximum absorbance peak values at 304 nm by DUV.Hydrostatic pressure (7.9 MPa) can promote the conjugation at certain pHvalues (e.g., at 7.0), but it can suppress the conjugation at other pHvalues (e.g., at 6.5).

Methods to limit the Maillard reaction to the very initial stage, theformation of the Schiff base, are also provided. Controlling the extentof the Maillard reaction has the advantage of limiting the formation ofunwanted intermediates or advanced products. Using the present methodsit is possible to obtain a simple product that is comprisedsubstantially of Schiff base with white color and with relatively highthermal stability (denaturation temperature 88° C.) in comparison to thenative whey protein (denaturation temperature 74° C.). Thus, in oneaspect, the compositions of the present invention remain at the Schiffbase level. Measurable amounts of Schiff base can be obtained relativelyrapidly, within about 1.5-2 hours of the beginning of the reaction.

In comparison to existing methods, the methods of the present inventionoffer improved protection against protein aggregation, allowing for theuse of higher treatment temperatures. The methods of the presentinvention require less time and energy than the dry heating method. Theresulting purified protein-polysaccharide conjugate obtained accordingto present invention exhibits improved thermal stability, more desirablecolor, and excellent emulsifying properties that are superior to boththe unmodified whey protein and the gum arabic. Some comparativeadvantages of the novel methods in accordance with the present inventionare outlined in Table 1 below. In Table 1, PS refers to polysaccharides;PPC refers to protein-polysaccharide conjugates.

TABLE 1 Some comparative advantages of the present invention (wet-heattreatment) relative to the conventional dry-heat treatment methodWET-HEAT TREATMENT CONVENTIONAL DRY- CONDITIONS (present invention) HEATTREATMENT Type of materials Aqueous solutions of both Dried powderedmixtures of needed for the protein and PS materials each materialreaction Humidity No specific humidity Maintain constant requirementsrequirement temperature (e.g., 80° C.) and relative humidity (e.g., 79%)Reaction time From 4 to 24 hours (60- 3-5 weeks (60° C.) and temperature70° C.) Type of products Products are almost Products are a complexformed in the completely composed of mixture of Intermediate andreactions Schiff base Advanced Maillard products Color of PPC orProducts are white in color Products have a yellow or reaction mixturebrown color Level of protein Little protein denaturation or Unknown howmuch protein denaturation aggregation occurs during denaturation occursduring reaction, stabilized by high reaction (not usually [PS]determined)

Some embodiments of the present invention are described in Zhu et al.,2008, J. Agric. Food Chem. 56: 7113-7118, which is herein incorporatedby reference.

EXAMPLES

It is to be understood that this invention is not limited to theparticular methodology, protocols, subjects, or reagents described, andas such may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention, which islimited only by the claims. The following examples are offered toillustrate, but not to limit the claimed invention.

Materials

Whey Protein Isolate (WPI) was provided by Davisco Foods International,Inc. The total amount of protein in the dry powder was >95% and thelactose was low (<1%). Before use, WPI was dissolved in Milli-Q waterand thoroughly dialyzed against Milli-Q water (dialysis membrane tubinghad a molecular weight cut-off of 6,000-8,000 Da) for 3 days at 5° C.with changes in water every 6 h to remove lactose. After lyophilization,purified WPI was stored at 5° C. prior to its use.

Dextran (Batch #115k0686, CAS 9004-54-0) from Leuconostoc mesenteroides,with molecular weight 8,500-11,500 (11 kDa), was obtained fromSigma-Aldrich (St. Louis, Mo.). Before use, dextran was dissolved inMilli-Q water and dialyzed against Milli-Q water for two days at 5° C.with changes in water every six hours. The dialysis membrane tubing hada molecular weight cut-off of 3,000 Da (Fisher Scientific, Pittsburgh,Pa.). After lyophilization, purified dextran was stored at 5° C.

Prestained SDS-PAGE molecular weight standards (Broad range) werepurchased from Bio-Rad Laboratories (Hercules, Calif.). The GelCodeglycoprotein staining kit was purchased from Pierce Biotechnology(Rockford, Ill.).

Preparation of Conjugates by Heat Treatment

Mixtures of WPI-dextran in various ratios (by weight) were dissolved in10 mM sodium phosphate buffer (NaPi) solution (pH 6.8). Sodium azide(0.02%, w/w) was added to prevent bacterial growth. The sample solutionswere stirred on a magnetic stirrer at room temperature (˜22° C.) for 2 hto completely dissolve the mixture. The pH values of the solutions wereadjusted by carefully adding 0.1 N HCl or 0.1 N NaOH to the desired pH.Solutions were gently stirred overnight at 5° C. to ensure the completehydration of the macromolecules. Aliquots of the solutions (1.0 ml,dispensed into 1.5 ml eppendorf tubes) were placed in a water bathheated at various temperatures for 24 h. Samples were then taken out ofthe water-bath and immediately cooled down at ice-water bath.Triplicates were carried out for each experiment.

Time Course of the Reaction

Using the same heat treatment for the sample preparation, a number ofsample solutions were incubated at 60° C. for different time periods.The sample solutions typically included WPI-dextran, and more typically10% WPI-30% dextran, at pH 6.5. At each time point, three sample tubeswere taken out from the water bath and immediately cooled down atice-water bath before analysis. Triplicate experiments were performed.

Formation of Conjugates Under Hydrostatic Pressure Treatment

Sample solutions of WPI-dextran (12 ml) mixtures prepared as describedabove were transferred into a pressure cell of a rheometer (UniversalDynamic Spectrometer, Paar Physica UDS 200 Physica Messtechnik GmbH,Stuttgart, Germany). Pressurization (up to maximum of 7.9 MPa) wasachieved by connecting the cell to a cylinder of compressed nitrogengas. No shearing was applied. Temperature and time were controlled bythe software attached to the rheometer. Three replicates were performedfor each sample; and three measurements were done for each replicate.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed on a Mini-Protean 3Cell (Bio-Rad Laboratories) bythe method of Laemmli (Laemmli, 1970, Nature 227: 680-685). Non-reducingand reducing SDS-PAGE analyses were carried out on a ready gel (TRIS-HClGel, 4-20% linear gradient, 15 well, Bio-Rad Laboratories). Samplesolution (15 μl corresponding to 40 μg of whey protein) was loaded intoeach well. Electrophoresis was run for 35 min at 200 V in 0.025 MTris-HCl buffer solution (pH 8.3, including 0.192 M glycine and 0.1%SDS, w/w) at room temperature. Two gels were run at the same time. Afterelectrophoresis, one gel was stained for protein by Coomassie blue G-250and the other one was stained for carbohydrate by the GelCodeGlycoprotein staining kit (Pierce Biotechnology), respectively. Theprotein stain was destained with 10% acetic acid (v/v) containing 30%methanol (v/v).

Difference UV Spectroscopy (DUV)

DUV measurements were carried out on a UV-visible spectrophotometer(Shimadzu UV-1601 PC, Shimadzu Corporation, Kyoto, Japan), in a 1-cmquartz cell at a constant temperature of 20° C. The samples were diluted33.3-fold, followed by centrifugation at 16,000×g for 10 min in anEppendorf Centrifuge (Model 5414, Brinkmann Instruments Inc., Westbury,N.Y.) at room temperature. The supernatant was used for a wavelengthscan from 270 to 500 nm. The difference absorption spectra of conjugatesafter processing were recorded against an un-reacted sample, which wasused as a reference. The extent of conjugation was assessed by the DUVabsorbance peak value at about 304 nm (Blonski et al., 1997, Biochem. J.323: 71-77).

Appearance of WPI-Dextran Conjugates

When solutions of 10% WPI-30% dextran (pH 6.5) were incubated at 60° C.for 24 h, the solutions in the eppendorf tube appeared white and opaque.There was no obvious aroma or smell, nor was any significant change inviscosity observed within the 24 h incubation period. When the samplewas centrifuged at 16,000×g for 30 min, the supernatant appeared clear,translucent, and the precipitate was white. The supernatant, where theconjugate resided, was typically used for the analyses described herein.

To determine if the precipitate was protein or dextran, aliquots of theprecipitate were subjected to several tests. The precipitate hardlydissolved in 8 M urea or 10% SDS either in the absence or presence of2-mercaptoethanol. These observations made it unlikely that theprecipitate was denatured WPI proteins. Dextran self-associates insufficiently concentrated solutions, and these dextran associates can bedissolved when re-suspended in distilled water and autoclaved for 30 min(Cadwallader et al., 1958, J. Amer. Pharm. Assoc. 47: 894-895). When theobtained white precipitate was re-suspended in Milli-Q water (4-10%),the precipitate apparently dissolved when heated at 100° C. for 30 min.This indicated that the white precipitate was due to the association ofdextran.

With prolonged incubation time (>24 h), a strong aroma was noted, and ayellow color appeared. The reaction was typically stopped after 24 hincubation by rapidly cooling down the samples in an ice-water bath.

WPI-Dextran Conjugate Interaction Studied by SDS-PAGE

The reaction between WPI and dextran was investigated by SDS-PAGE. Asshown in FIG. 1 (protein stain), under non-reducing conditions (lanes1-4 in FIG. 1A), unheated WPI (native WPI, lane 1, FIG. 1A) and themixture of 10% WPI-30% dextran (5° C. for 24 h, lane 2, FIG. 1A) hadidentical and characteristic bands of WPI. As identified in FIG. 1, twomajor bands were assigned to monomers of α-La and β-Lg respectively; twominor bands were attributed to dimers of β-Lg and BSA; and the fourfaint bands were LF, IgG, IgL and IgH. This indicated that nopolymerization occurred in WPI in the presence of 30% dextran withoutheat treatment of the mixture. For the heat-treated 10% WPI alone (lane3, FIG. 1A), the α-La, β-Lg dimer, BSA and IgG bands were significantlydiminished; the intensity of the β-Lg monomer band was reduced;meanwhile a dense band appeared on the top of the gel. This indicatedthat proteins in WPI had associated into large molecular polymers thatcould not migrate into the separating gel. In the case of heat-treatedmixture of 10% WPI-30% dextran (lane 4, FIG. 1A), a new diffuse bandthat had a molecular weight distribution of 26-98 kDa appeared in theseparating gel, indicating the formation of new protein species, whichmigrated into the separating gel. In addition, a thin band of largemolecular weight polymers was observed on the top of the gel similar tothe heated 10% WPI sample. Compared to the heated 10% WPI sample alone(lane 3, FIG. 1A), the intensity of the band with large molecular weightpolymers was less intense in the heated mixture of 10% WPI-30% dextran(lane 4, FIG. 1A). This indicated that the polymerization of WPI wasgreatly inhibited in the presence of 30% dextran. A similar phenomenonwas reported by Schmitt et al., 2005, J. Agric. Food Chem. 53:9089-9099, where the polymerization of β-lactoglobulin was greatlyreduced by the presence of acacia gum. Garrett et al., 1998, J. DairySci. 71: 10-16, suggested that the inhibition of the thermal aggregationof whey proteins by various sugars was caused by reducing hydrophobicintermolecular interactions between protein molecules.

Since non-covalent interactions were dispersed during SDS-PAGE, the newprotein species and the large molecular weight polymers on the top ofthe gel in lane 4 (FIG. 1A) were linked by some types of covalent bonds.Under reducing conditions, for lanes 7 and 8 (FIG. 1A), the largemolecular weight polymers which resided at the top of lanes 3 and 4(FIG. 1A) completely disappeared, indicating that these large polymerswere linked by disulfide bonds. The heated 10% WPI under reducingconditions (lane 7, FIG. 1A) had identical bands compared to native WPI(unheated, lanes 5 and 6, FIG. 1A), while the heated mixture of 10%WPI-30% dextran still had the diffuse band with the molecular weightdistribution of 28-100 kDa (lane 8, FIG. 1A) that appeared at the samelocation as the new band in lane 4 (FIG. 1A). Since the new proteinspecies were observed in both lanes 4 and 8 (FIG. 1A), this indicatedthat the species were linked by covalent bonding other than disulfide.The slightly larger molecular weight of the bands in the presence of2-mercaptoethanol was due to the larger hydrodynamic size of the S—Sreduced proteins.

SDS-PAGE was also performed with carbohydrate staining using thePeriodic Acid-Schiff reagent (PAS) (FIG. 1B). No bands were observed forlanes 1-3 and 5-7. This is because protein bands are not stained by PAS.Being a neutral molecule, dextran could not migrate into the separatinggel. Similar to FIG. 1A, a faint high molecular weight band was observedat the top of the gel in lane 4 (FIG. 1B) and it disappeared in lane 8(FIG. 1B), indicating that the polymers (lane 4) were reduced into smallmolecules in the presence of 2-mercaptoethanol (lane 8). The same broaddiffuse bands as in the lanes 4 and 8 (FIG. 1A) were also observed inthe separating gel in lanes 4 and 8 of FIG. 1B, corresponding to amolecular weight distribution of 26-98 kDa (lanes 4, FIG. 1B), and28-100 kDa (lanes 8, FIG. 1B), respectively. The above results confirmedthat glycosylated protein species, such as WPI-dextran conjugates, wereformed in the mixture of 10% WPI-30% dextran as a result of heating at60° C. for 24 h. The molecular weight distribution of the conjugates was26-98 kDa. Some conjugates were also linked through disulfide bonding ofproteins.

The broad molecular weight distribution of the conjugate bands was dueto the nature of the components of the two reactants. WPI consists ofseveral protein types, such as α-La (˜18%), β-Lg (˜52%), BSA and IgG(5%) (Wang and Lucey, 2003, J. Dairy Sci. 86: 3091-3101). Each proteincontains multiple potential reactive sites. For example, β-Lg contains16 potential reactive primary amino groups, which are one α-NH₂ group atthe N-terminal and fifteen ε-NH₂ of the lysine residues. From theintensity of the remaining bands of WPI proteins (FIG. 1A), it appearedthat each of the WPI proteins was partly involved in the conjugation ofWPI-dextran to different extents. The molecular weight of dextran variesfrom 8,500-11,500 Da as indicated by the supplier (Sigma). Thepolydispersity of dextran also led to a broad molecular weightdistribution of the WPI-dextran conjugates.

Confirmation of Schiff Base Formation in WPI-Dextran Conjugates by DUV

In order to further clarify the nature of the covalent bonding inWPI-dextran conjugates, DUV spectra were recorded by scanning wavelengthbetween 270-500 nm. As shown in FIG. 2, for the heated mixture of 10%WPI-30% dextran solution, the interaction of WPI-dextran resulted in aDUV spectrum characterized by a maximum at 304 nm, which was a clearindication of Schiff-base formation (Heinert and Martell, 1963, J. Am.Chem. Soc. 85: 183-188; Blonski et al., 1997, Biochem. J. 323: 71-77).No change in absorbance was observed for a heated 30% dextran solutionover all the scanned wavelengths. A small absorbance peak at 304 nm wasobserved for the heated 10% WPI alone. This was probably caused by somelow concentration of residual lactose in WPI (even though it wasextensively dialyzed). The DUV measurements shown in FIG. 2 wereperformed after 33.3-fold dilution of the original samples.

As indicated in FIG. 2, the DUV absorbance peak of the WPI-dextranSchiff base was asymmetric and tailed into the visible wavelengths. Thiscould be explained by the differences in the environment of the Schiffbase, which results in a broader absorbance peak. It was also possiblethat the simultaneous formation of many chromophores led to the tailingof the peak as reported in other studies (Hofmann, 1998, J. Agric FoodChem. 46: 3891-3895). The conjugates of WPI-dextran mainly consisted ofthe Schiff base due to formation of this peak at 304 nm. The DUVabsorbance value at 304 nm was used to estimate the extent of theconjugation.

Storage of the Schiff base (without dilution at −80° C. for at leastthree months; or a diluted, 33.3 fold, solution at 5° C. for at least 1week) did not result in any change in absorbance value at 304 nm,indicating that the Schiff base of WPI-dextran conjugates was stable.Not wanting to be bound by the following explanation, this stability wasprobably due to the water restricted environment of the Schiff base C═Nbond, thus preventing a H₂O molecule from hydrolyzing the C═N bond.

With prolonged incubation of mixtures of 10% WPI-30% dextran (60° C. for48 h), the absorbance peak slightly red-shifted to ˜310 nm; meanwhile asmall new shoulder appeared around 355 nm. The resultant solution waslight yellow. This implied that the WPI-dextran conjugates/Schiff basehad developed into the intermediate stage of the Maillard reaction.

Time Course of the Conjugation Reaction

The formation of the Schiff base from a WPI-dextran mixture was studiedas a function of time. As shown in FIG. 3, the conjugation reaction wastime-dependent. The formation of the conjugates/Schiff base graduallyincreased with time during the first 24 h when incubated at 60° C. Therewas little further increase between 24-48 h of incubation at 60° C.(FIG. 3). The measurements shown in FIG. 3 were performed after33.3-fold dilution of the original samples. SDS-PAGE under reducingconditions and with carbohydrate staining indicated that the molecularweight distribution of the conjugates increased with time from 28-36 kDa(2 h at 60° C.); 28-52 kDa (4 h at 60° C.); 28-70 kDa (6 h at 60° C.);28-82 kDa (8 h at 60° C.); 28-95 kDa (12 h at 60° C.); and 28-100 kDa(24 h at 60° C.). The intensity of the bands, particularly the lowermolecular weight species increased with time. This means that thesmaller molecular weight proteins in WPI (α-La, β-Lg) reacted morerapidly with dextran than the large molecular proteins (BSA, IgG), andit was in the order α-La>β-Lg>BSA and IgG. This observation was inagreement with the results of Nacka et al., 1998, J. Protein Chem. 17:495-503, who reported that the glycosylation with reducing sugars withα-La was faster than with β-Lg, based on SDS-PAGE. The higherconcentration of α-La and β-Lg in WPI compared to BSA and IgG was alsoresponsible for the faster conjugation rate of α-La and β-Lg withdextran.

A reaction time of 24 h at 60° C. was chosen under the experimentalconditions to allow the reaction to proceed with the highest yield ofSchiff base but with low levels of intermediate and advanced Maillardproducts.

Effect of WPI and Dextran Concentration on Schiff Base Formation

The effect of WPI or dextran concentration on the conjugation ofWPI-dextran was examined. As shown in FIG. 4, the formation ofWPI-dextran Schiff base increased almost linearly with increasing WPIconcentration from 2.5 to 10% in a mixture with 30% dextran. Furtherincreases in the WPI concentration above 10% led to the gelation of WPIduring heating at 60° C. Each protein in WPI has multiple potentialreactive amine groups. However, at pH 6.5, most of the ε-amino groups(>99%) are in the protonated form, which are not reactive. Themeasurements shown in FIG. 4 were performed after 33.3-fold dilution ofthe original samples.

The effective concentration of —NH₂ groups in the system is lower thanthe concentration of carbonyl group. Therefore, the conjugationincreases almost linearly with WPI concentration. In FIG. 5, theformation of WPI-dextran Schiff base increased with the increase indextran concentration from 10 to 40%, in mixtures with 10% WPI. Themeasurements shown in FIG. 5 were performed after 33.3-fold dilution ofthe original samples. Dickinson and Semenova (1992, Colloids Surf. 64:299-310) thought that each polysaccharide molecule (dextran) has onlyone reducing group capable of reacting with amine groups in proteins,and thus the extent of conjugate formation increases with an increase inthe proportion of polysaccharide. This is also true for protein aminogroups at pH 6.5 since more than 99% of the amino groups are in thenon-reactive NH₃ ⁺ form. The fitting-equation in FIG. 5 indicated thatthe conjugation degree was positively related to the logarithm of theconcentration of dextran. This might be related to the bulk structure ofdextran that restricts its access to the reactive sites of the aminogroups on proteins.

Increasing the dextran concentration from 30 to 40% did not result insignificant increase in the WPI-dextran Schiff base formation (FIG. 5),but a marked increase in viscosity was observed. It is possible that thehigh viscosity prevented the accessibility of the reducing end ofdextran to WPI proteins due to the overlapping and interpenetration ofthe coil structure of dextran molecules with themselves at very highconcentrations; 10% WPI and 30% dextran were therefore chosen for theconjugate formation.

An excess of polysaccharide compared to protein in the mixture ofreactants (1:3-1:9, by weight) is often used in preparing Maillardreaction products by dry-heating. There is a lack of detailedexplanation for this ratio. One plausible explanation was that eachpolysaccharide molecule has only one reducing group capable of reactingwith amine groups in the proteins, thus the extent of conjugateformation would increase with increasing proportion of polysaccharide(Dickinson and Semenova, 1992, Colloids Surf. 64: 299-310). However, forthe reaction in aqueous solutions, the mixing ratio of reactants did notmean it was the binding ratio of protein to polysaccharides. For theconjugates formed at 10% WPI-30% dextran (pH 6.5, 60° C. for 24 h), thecompositional analysis of the individual components indicated that only1.9% (by weight) WPI and 5.6% (by weight) dextran were involved in theformation of WPI-dextran Schiff base. Most dextran molecules remainedunreacted. Not wanting to be bound by the following explanation, thiscould be the consequence of a crowding effect, due to the high dextranconcentration in the mixture of 10% WPI-30% dextran, that led to anincreased probability of collisions between amino groups on WPI proteinsand carbonyl groups on dextran, thus promoting the conjugation reaction(Ralston, 1990, J. Chem. Educ. 67: 857-860; Minton, 1997, Curr. Opin.Biotech. 8: 65-69; Somalinga and Roy, 2002, J. Biol. Chem. 227:43253-43261). The association of WPI, which might be also enhanced bycrowding effect, was offset by the protective effect of dextran, asdiscussed previously.

Effect of Temperature and pH

Heating a mixture of WPI and dextran solution promotes two competitivereactions—aggregation of WPI proteins and conjugation between WPI anddextran. High temperature favors the conjugation reaction, as chemicalreactions are favored by an increase in temperature. High temperaturealso promotes WPI denaturation/aggregation, thus leading to the loss ofreactant for the conjugation of WPI-dextran. The critical structuralchanges in β-Ig occur at 63° C. and pH 7.0 (from circular dichroismspectroscopy; Prabakaran and Damodaran, 1997, J. Agric. Food Chem. 45:4303-4308); the denaturation temperatures of α-La is 64° C. at pH 7.0(from differential scanning calorimetry; McGuffey et al., 2005, J.Agric. Food Chem. 53: 3182-3190); and BSA is 62.2° C. at pH 6.7(Mulvihill and Donovan, 1987, J. Food Sci. Tech. 11: 43-75). Therefore,the effect of temperature on the conjugation of WPI-dextran wasinvestigated at <60° C. in order to minimize denaturation/aggregation ofWPI proteins. Dextran is stable at ambient temperatures over the rangeof pH 4-10. Any structural change in dextran during heat processing wasignored in this study. As shown in FIG. 6, the Schiff base was hardlyformed at low temperatures (40 or 50° C.) over the pH range studied.Regardless of pH, the formation of Schiff base was favored by raisingthe temperature.

As far as pH was concerned, as seen in FIG. 6, the effect of pH on theSchiff base formation was negligible at lower temperatures (40° C. and50° C.). At 60° C., the formation of Schiff base was significantlyenhanced by reducing the pH from 7.0 to 6.5. There was no significantdifference in the reaction in the pH range 7.0 to 8.5. The generation ofSchiff base was greatly enhanced at pH 6.5. This was in good agreementwith the literature (Ames, 1990, Trends Food Sci. Tech. 1: 150-154). Inone example, the conditions pH 6.5 and 60° C. were used to maximize theextent of formation of Schiff base in the study. The measurements shownin FIG. 6 were performed after 33.3-fold dilution of the originalsamples.

Effect of Hydrostatic Pressure on the Schiff Base Formation

To examine if the formation of WPI-dextran conjugate/Schiff base wouldbe facilitated by applying elevated pressure, hydrostatic pressure (7.9MPa) was used in combination with heating during incubation. For the WPIproteins, the pressure required to unfold the proteins has been reportedto be 50, 200 and 800 MPa for β-Lg, α-La and BSA, respectively(López-Fandiño, 2006, Crit. Rev. Food Sci. Nutr. 46: 351-363).Considering the protective effect of polysaccharides on proteins againstelevated pressure (Galazka et al., 1999, Food Hydrocoll. 13: 81-88), theconformational change of WPI proteins under 7.9 MPa pressure could beneglected. Assuming that the structure of dextran was not affected bythe applied pressure, any pressure-induced difference was assumed to bedue to an impact on the conjugation reaction.

As shown in FIG. 7, the amount of Schiff base at 7.9 MPa was marginallyincreased by reducing the pH from 8.5 to 8.0 compared with the reactionat atmospheric pressure. Schiff base formation was significantlyinhibited by the application of pressure at pH 6.5, but significantlyenhanced at pH 7.0 compared to at atmospheric pressure. The lack ofinfluence of pressure could be due to the low pressures used in thisstudy.

There have been various reports about the impact of pressure on theinitial step of the Maillard reaction. For example, high pressure(50-500 MPa, 50° C., pH 8.2) hardly affected the initial condensationreactions (Tamaoka et al., 1991, Agric. Biol. Chem. 55: 2071-2074).However, high pressure (60 MPa, at 70° C.) was reported to acceleratethe initial reactions at pH 7.0 due to the negative activation volume(Isaacs and Coulson, 1996, J. Phys. Org. Chem. 9: 639-644). Theseobservations were in accordance with the results herein; that is, theeffect of pressure was related to the pH of solution. Moreover, Morenoet al., 2003, J. Agric. Food Chem. 51: 394-400) reported that at 400 MPa(60° C., pH 5-8) Amadori rearrangement products were not appreciablyaffected by pressure in unbuffered media but were suppressed in bufferedmedia. It was attributed to the pressure-induced dissociation of theacid groups. It was possible that, due to the pressure-induceddissociation of the acid groups, high pressure accelerated thecondensation reaction of ε-amino to carbonyl at pH ≥7.0; but it retardedthe formation of the adduct due to the protonation of the ε-amino groupat pH <7.0.

Emulsifying Properties of Protein-Polysaccharide Conjugates

In general, PPC exhibit better functional properties than proteins andPS alone. It is likely (but not certain) that this improvement can beattributed to the structure and size of the conjugate (Chevalier et al.,2001, Int Dairy J. 11: 145-152). Thus, the functional properties of PPCare of interest especially for their viscosity, gelation and emulsifyingabilities. For example, a superior biopolymer for use in oil-in-wateremulsions would be a PPC that combines the surface-active properties ofthe protein with the potential steric-stabilizing properties of theassociated PS (Dickinson and Semenova, 1992, Colloids Surf. 64:299-310). The hydrophilic nature of the PS should allow it to extendinto the aqueous phase.

In one example, preparation of emulsions from purified PPC prepared fromthe conjugation of WPI with dextran (500 kDa). FIG. 8 shows particlesize distribution (d32) of emulsions during storage that were preparedwith 0.5% (wt) of each emulsifying material, 20% (v/v) soybean oil, 10mM NaPi buffer, pH 6.5, 0.02% NaN₃. Emulsions were prepared with anAvestin EmulsiFlex high pressure homogenizer (1.5 kilobar). Samples werestored at room temperature for at least 6 weeks. The performance of thePPC made in accordance with the present invention was compared with gumarabic as well as WPI and mixtures of WPI and dextran, Dx. The producedPPC contains about 10% protein, i.e. 0.05% protein for the PPC sample.All emulsions were stabilized with 0.5% (wt/wt) of emulsifying material.Even with the low level of protein the novel emulsions stabilized withPPC were not significantly different from those stabilized with 0.5%WPI. This indicated that the produced PPC was an excellent emulsifierand superior to gum arabic. Simple mixtures of WPI with dextran (notconjugated) did not produce stable emulsions since the protein level wasvery low (0.05%) in these samples.

Solubility of the Protein-Polysaccharide Conjugates

FIG. 9 is a graph showing the solubility of conjugates (0.1% protein) atroom temperature and heated 30 min at 80° C. within the pH range3.5-7.5, in comparison with whey protein (0.1% WPI). The higher theabsorbance at 500 nm, the lower the solubility is.

FIG. 10 is a graph showing the solubility of conjugates (0.1% protein,pH 4.5) at room temperature and heated 30 min at 80° C., in comparisonwith whey protein (0.1% WPI). The higher the absorbance at 500 nm, thelower the solubility is.

Thermal Stability of the Protein-Polysaccharide Conjugates

FIG. 11 is a graph showing thermal stability of the obtained conjugates.A DSC heating scan (1.0° C./min) for 10% conjugate (dotted line) and 10%WPI (solid line) at pH 8.5 is shown. The conjugates had greatly improvedthermal stability.

Effect of Solids Content of a Mixture of Sodium Caseinate

FIG. 12 is a graph showing the effect of solids content of a mixture ofsodium caseinate: maltodextrin (1:1 ratio) on the formation of Schiffbase (as indicated by the ΔA305 nm or change in DUV at 305 nm) and color(ΔA450 nm). Samples were heated for 90 min at 95° C. and pH 6.5Maltodextrin was 250 DE. Different letters indicate significantdifferences (P<0.05). This demonstrates that other types of proteins andpolysaccharides (apart from, or in addition to, whey protein anddextran) can successfully undergo this reaction.

Measurement of Emulsion Stability by Whey Protein-Dextran Conjugates andthe Stability of Emulsions After Heat Treatment and in Low pH/High SaltSystems

Radiolabeling of Protein-Polysaccharide Conjugate.

The conjugate is radiolabeled with [¹⁴C] nuclide by reductivemethylation of amino groups with [¹⁴C]-formaldehyde. Briefly, 60 μl of[¹⁴C]-formaldehyde solution (containing 0.01 mmol of formaldehyde(having a total radioactivity of 0.1 mCi) is mixed with 40 ml of 20 mMphosphate buffer containing 40 mg of protein, followed by addition of 50mg of NaCNBH₃. After incubation for 2 h at room temperature, thereaction mixture is dialyzed exhaustively against pure water (surfacetension 72.9 dynes/cm at 20° C.) and lyophilized in cryo-vials andstored frozen at −70° C. This labeling protocol usually results in [¹⁴C]labeling of about 1-2 amino group per protein molecule.

Adsorption at the Oil-Water Interface.

Adsorption of PPC from the aqueous phase to the triolein-water interfaceis studied by the surface radiotracer method. A brief description ofthis method is as follows. The method essentially involves spreading ofa 100 nm thick triolein layer on water surface and monitoring adsorptionof ¹⁴C-radiolabeled proteins using a surface radiotracer probe. Thesurface tension is measured by the Wilhelmy plate technique using a ST9000 surface Tensiometer (Nima Technology Ltd., Coventry, England),interfaced with an IBM PC. The apparatus consists of teflon troughhaving inner dimensions of 17.45×5.5×4 cm³. One side of the trough has asmall hole (1 mm diameter) capped tightly with a septum for injectingthe protein solution into the bulk phase. In each experiment, 350 mL ofa solution consisting of 20 mM phosphate buffer (pH 7.0) adjusted to 0.1M ionic strength with NaCl is used as the bulk phase. Prior to spreadingthe triolein film over the buffer surface, a very thin (3 mm dia, 12.7mm length). Teflon-coated magnetic stir bar is placed at the center ofthe trough and the radiotracer probe (Ludlum Measurements, Inc.,Sweetwater, Tex.) and the Wilhelmy plate is placed in position. Themethod for spreading a 1000 nm thick triolein film has been described indetail elsewhere (Sengupta and Damodaran, 1998, J. Colloid InterfaceSci. 206: 407-415).

To initiate protein adsorption, a known volume (0.5-2.0 ml) of theradio-labeled protein/PPC stock solution is injected through the septumon the side of the trough. The final concentration of protein in thebulk solution is in the range of 1-10 μg mL⁻¹ (10⁻⁴-10⁻³% w/v). Thesurface tension and surface radioactivity (cpm) measurements areinitiated soon after injecting the protein solution. The bulk phase isstirred gently by using the stir bar at low speed (60 rpm), which doesnot cause ripples on the triolein film. The bulk phase is stirred onlyfor the first 15 minutes.

The surface tension and surface cpm is continuously monitored until theyreach an equilibrium value, which usually takes about 20-24 h. In theseexperiments, the attainment of equilibrium is defined as the conditionwhen the surface cpm does not change for at least 4 h. The cpm isrecorded using a rate meter (Model 2200, Ludlum Measurements,Sweetwater, Tex.) and printed out on a strip chart recorder interfacedwith the rate meter. The cpm measurements are made at 1 min intervalsfor the first hour of the experiment, followed by measurements at 10 minintervals thereafter. Calibration curves required for converting cpmreadings at the oil-water interface into interfacial proteinconcentrations (mg m⁻²) are constructed as described elsewhere (Senguptaand Damodaran, 1998, J. Colloid Interface Sci. 206: 407-415).

The cpm versus interfacial radioactivity (μCi m⁻²) calibration curve isconstructed by spreading ¹⁴C-labeled β-casein at the oil-waterinterface. The cpm arising from radioactivity of protein in the bulksolution is determined from a standard curve relating interfacial cpmversus bulk radioactivity of Na₂ ¹⁴CO₃. The interfacial radioactivity(μCi m⁻²) is determined by dividing the background-corrected cpm withthe slope of the cpm versus interfacial radioactivity calibration curve.The interfacial protein concentration (mg m⁻²) is obtained by dividingthe instantaneous interfacial radioactivity (μCi m⁻²) with the specificradioactivity of the protein (μCi mg⁻¹).

Experiments may be conducted on both native protein and PPC to elucidatethe effect of the PS moiety on the rate and extent of adsorption as wellas on the final surface tension values.

Emulsifying Properties. To develop a basic understanding of the effectof the PS moiety on interfacial properties of proteins, the emulsifyingproperties of PPC conjugates are examined and compared to pure proteins.Emulsions may be prepared by homogenizing 1% protein or PPC solutions in20 mM imidazole-HCl buffer, pH 7.0, containing 0.02% sodium azide, withsoybean oil. The volume fraction of oil in all emulsions is set at 20%v/v. A coarse emulsion of the protein solution and oil mixture ishomogenized (two passes) in an Emulsiflex-B3 (Avestin Inc., Ontario,Canada) homogenizer at an input pressure of 200 kPa, corresponding to apressure drop of 40 MPa. This provides an emulsion with about 1 μmaverage particle size distribution. The kinetic stability of theemulsion over a period of 30 days at 25° C. is studied by measuring rateof change of emulsion droplet size using the Malvern Mastersizer.Specifically, changes in the size distribution and total interfacialarea of the emulsion as a function of storage time is determined foremulsions made with PPC and pure proteins. The impact of processingconditions is tested, including various heat treatments, salts and pHvalues on the properties of the obtained PPC-stabilized emulsions andthese are compared to WPI stabilized emulsions.

Measurement of the Astringency of Low pH Beverages Made with WheyProtein-Dextran Conjugates

Astringency in protein or PPC fortified beverages is determined usingtrained sensory panels. Tannic acid solutions at 0, 0.38, 0.60, 0.93,1.45, and 2.26 mM are used as astringency standard solutions correlatingwith astringency scores of 0, 2, 4, 6, 8, and 10, respectively. Theastringency of the protein sample is evaluated by astringency scorecompared with the astringency of standard tannic acid solutions. A 10 or15-point scale is used for the sensory panel (Beecher et al., 2006).Panelists are pre-screened for their ability to correctly detectastringency and differentiate it from other attributes, such asbitterness or sourness. The rate of build-up of astringency duringsample evaluation is determined. When the beverage is formulated, onemay add enough PPC to achieve between 3 to 5% protein addition. Thebeverage is adjusted to pH values between 3 to 4.5 and a neutral pHbeverage is also examined. 2.0 M HCl or NaOH is used for pH adjustment(Sano et al., 2005, J. Dairy Sci. 88: 2312-2317). Protein fortifiedbeverages are dialyzed against 5 mM sodium phosphate buffer, already atthe desired pH (to have a similar ionic strength in the beverages).Beverages are either unheated or heated at 85° C. for 10 min. A controlbeverage is also prepared with unmodified protein (we will use severaldifferent commercial brands of WPI). All the beverages are adjusted tothe same viscosities (or flow behavior) using added gums. A skilledartisan will know to check that the gums themselves do not alter theastringency of beverages.

The impact of the degree of conjugation and the molecular weight of thePS used for conjugation on the astringency of beverages may be examined.It is possible that there is a minimum molecular weight of dextran thatis able to confer the ability to reduce the astringency reaction. Theimpact of the concentration of added PPC on the astringency of beveragesmay also be tested. Compared to unmodified whey protein, it may bepossible to add a higher concentration of the PPC before exceeding anacceptable astringency threshold (as determined from sensory panelists).Alternatively, one may instead switch to the use of maltodextrin as thePS. In some embodiments, it is possible to conjugate whey proteins withthis PS using the methods of the present invention. In addition, one maydirectly examine the interactions between SPRPs and the PPC of thepresent invention using the turbidity method of Horne et al., 2002,Chem. Senses 27: 653-659. The PPC of the present invention may havegreatly reduced turbidity compared to the native protein even added atthe same concentration.

Conjugation of Whey Proteins or Whey Protein Hydrolysates May ReduceMilk Allergenicity

WPI, pure β-Ig (Sigma), and whey proteins hydrolyzed to various extents(DH values) may be conjugated in these experiments. Before use, WPI,β-Ig and whey hydrolyzates are dissolved in Milli-Q water and thoroughlydialyzed against Milli-Q water (dialysis membrane tubing molecularweight cut-off 6,000-8,000 Da) to remove free lactose for 3 days at 5°C. with changes in water every 6 hours. After lyophilization, purifiedproteins are stored at 5° C. for use. Commercial infant formula thatcontains partially hydrolyzed whey, e.g. Good Start Milk Based InfantFormula Powder (Nestle), also may be used.

The impact of the molecular weight of PS (10-500 kDa) on allergenicitymay be examined. Various methods for detecting milk allergens exist. Invitro tests that measure the capacity of specific IgE from serum ofallergic patients to bind the modified protein(s) may be used. Sera of anumber (e.g. at least 20) patients with a history of reactions to cow'smilk are obtained. Approximately 10 ml is obtained from these patientsfor allergenicity testing. Serum of a subject without CMA is used as acontrol. Allergenicity is determined using specific ELISA immunoassaysfor whey proteins (commercially available, e.g. Neogen and R-Biopharm)and allergen specific IgE methods such as Western blotting and RAST(Hamilton and Adkinson, 1983; J. Clinical Immunoassay 6: 147-153).Purified PPC is used as the allergen in IgE enzyme-linked immunosorbentassay (ELISA). Most ELISAs are not successful in the analysis ofhydrolyzed protein, thus allergenicity may also be checked via IgEmethods such as Western blotting and radioallergosorbent test (RAST).

An example of the general test method for a commercial ELISA test formilk allergens from Neogen is as follows: ELISA microtiter plates (100μl/well) are used. PPC conjugate (various concentrations, e.g. 1-10μg/ml) is added to antibody-coated wells (capture antibody). Unboundresidue is washed away and a second, enzyme-labeled antibody (detectorantibody) is added. The detector antibody binds to the already boundPPC. After a second wash, the substrate is added (e.g. 100 μl of patientor control serum, diluted 1:10). Color develops as a result of thepresence of bound detector antibody. Red Stop reagent is added and thecolor of the resulting solution is observed. A microwell ELISA reader isused to yield optical densities. Control optical densities form astandard curve, and sample optical densities are plotted against thecurve to calculate the concentration of PPC.

Various types of commercially available kits may be used. Alternatively,methods similar to the one described by Hattori et al., 2004, J.Agricultural and Food Chemistry 52, 4546-4553, may be used.Alternatively, it may be possible to test the conjugate prepared frompurified β-Ig. PPC may reduce the antigenicity of β-Ig and wheyproteins. The reduction in the antigenicity of β-Ig by conjugation maydepend on the molecular weight of the PS as large PS could shieldepitopes.

A Western blotting technique similar to that reported by Matheu et al.,2004, Clinical and Molecular Allergy 2: 2, is used. SDS-PAGE isperformed with a 12% polyacrylamide gel and a stacking gel of 4% (thisis varied depending on the molecular weight of the PPC). About 5-20 μgof purified PPC are applied to every lane and electrophoresis isperformed (Mini Protean II System, Bio-Rad laboratories, Richmond, USA).Then, proteins are electrophoretically transferred from the separatinggel to Immobilon-P™ (PVDF, Millipore Corporation, Billerica, Mass., USA)membranes in a transfer buffer. After blocking with a solution ofgelatin 3% for 1 hour, the membranes are washed and incubated overnightwith patient's and normal control sera. Next day, membranes are washedand incubated with goat anti-human IgE-labelled-peroxidase. Detection isperformed with a chemiluminescence substrate (Pierce Chemical Company,Rockford, Ill.). The Western-blot should demonstrate if IgE in the CMApatient's serum is bound to some medium/high-molecular weight PPC bands.Control sera should be negative.

The allergenicity of each single protein is due to a number of molecularimmunoreactive structures, i.e. the IgE-binding epitopes that arewidespread within the protein molecule. The conjugation of whey proteinswith dextran molecules may interfere with the IgE-binding epitopes onthe protein molecule. In some embodiments, intact β-Ig or WPI may beused in reducing protein allergenicity. Alternatively, partiallyhydrolyzed whey proteins may be used as this product is only successfulwith some but not all patients with CMA; thus the conjugation ofpartially hydrolyzed whey may improve its hypoallergenicity. It isexpected that RAST analysis will indicate that IgE reactivity to the PPCof the present invention is at least several fold lower than that of theunmodified protein.

It is to be understood that this invention is not limited to theparticular devices, methodology, protocols, subjects, or reagentsdescribed, and as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which is limited only by the claims. Other suitablemodifications and adaptations of a variety of conditions and parameters,obvious to those skilled in the art of chemistry, biochemistry,molecular biology, and cheese manufacturing, are within the scope ofthis invention. All publications, patents, and patent applications citedherein are incorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A method of preparing a polysaccharide-proteinconjugate, comprising reacting a polysaccharide comprising a reducingsugar and a protein in an aqueous solution comprising 10-40% (w/v)polysaccharide and 1-30% (w/v) protein, under temperature conditions offrom about 40° C. to about 120° C., thereby producing apolysaccharide-protein conjugate, wherein the polysaccharide-proteinconjugate is substantially free of intermediate and advanced Maillardproducts and exhibits a maximum absorbance peak at a wavelength of lessthan 420 mm in difference ultraviolet spectroscopy scanned between 270nm and 500 nm.
 2. The method of claim 1, wherein the reacting isconducted at a pH of from about 6.0 to about 8.0.
 3. The method of claim1, wherein the reducing sugar and protein are reacted under temperatureconditions of from about 40° C. to about 120° C. for a period of fromabout 1 hour to about 48 hours.
 4. The method of claim 1, furthercomprising subjecting the solution to a hydrostatic pressure in theamount of 1-20 MPa.
 5. The method of claim 1 further comprising the stepof recovering the polysaccharide-protein conjugate from the solution. 6.A method of preparing a whey protein isolate-dextran conjugate,comprising reacting whey protein isolate and dextran in an aqueoussolution comprising 10-40% (w/v) dextran and 1-30% (w/v) whey proteinisolate, under temperature conditions of from about 40° C. to about 120°C., thereby producing whey protein isolate-dextran conjugate.
 7. Themethod of claim 6, wherein the solution is acidified to a pH of fromabout 6.0 to about 8.0.
 8. The method of claim 6, wherein the dextranand the whey protein isolate are reacted under temperature conditions offrom about 40° C. to about 120° C. for a period of from about 1 hour toabout 48 hours.
 9. The method of claim 6, further comprising subjectingthe solution to a hydrostatic pressure in the amount of 1-20 MPa. 10.The method of claim 6 further comprising the step of recovering the wheyprotein isolate-dextran conjugate from the solution.
 11. The method ofclaim 1, wherein an amount by weight of the polysaccharide in theaqueous solution is greater than an amount by weight of the protein inthe aqueous solution.
 12. The method of claim 1, wherein thepolysaccharide comprises at least one of galactomannan, maltodextrin,konjac mannan, xyloglucan, polydextrose, and dextrin.
 13. The method ofclaim 1, wherein the polysaccharide comprises maltodextrin.
 14. Themethod of claim 1, wherein the reducing sugar and protein are reactedunder temperature conditions of from 40° C. to 70° C. for a period offrom about 4 hours to less than 48 hours.
 15. The method of claim 1,wherein the reducing sugar and protein are reacted under temperatureconditions of from 40° C. to 70° C. for a period of from about 4 hoursto about 24 hours.
 16. The method of claim 1, wherein thepolysaccharide-protein conjugate exhibits a maximum absorbance peak at awavelength of less than 355 nm in difference ultraviolet spectroscopyscanned between wavelengths 270 nm and 500 nm.
 17. The method of claim1, wherein the polysaccharide-protein conjugate exhibits a maximumabsorbance peak at a wavelength of less than 310 nm in differenceultraviolet spectroscopy scanned between wavelengths 270 nm and 500 nm.18. The method of claim 1, wherein the polysaccharide-protein conjugateexhibits a maximum absorbance peak at a wavelength of about 304-305 nmin difference ultraviolet spectroscopy scanned between wavelengths 270nm and 500 nm.
 19. The method of claim 1, further comprising, after thereacting, cooling the aqueous solution to a temperature below thetemperature conditions to thereby yield the polysaccharide-proteinconjugate.
 20. The method of claim 19, wherein thepolysaccharide-protein conjugate exhibits a maximum absorbance peak at awavelength of less than 355 nm in difference ultraviolet spectroscopyscanned between wavelengths 270 nm and 500 nm.
 21. The method of claim19, wherein the polysaccharide-protein conjugate exhibits a maximumabsorbance peak at a wavelength of less than 310 nm in differenceultraviolet spectroscopy scanned between wavelengths 270 nm and 500 nm.22. The method of claim 19, wherein the polysaccharide-protein conjugateexhibits a maximum absorbance peak at a wavelength of about 304-305 nmin difference ultraviolet spectroscopy scanned between wavelengths 270nm and 500 nm.
 23. The method of claim 19, wherein the reducing sugarand protein are reacted under temperature conditions of from 40° C. to70° C. for a period of from about 4 hours to less than 48 hours.
 24. Themethod of claim 19, wherein the reducing sugar and protein are reactedunder temperature conditions of from 40° C. to 70° C. for a period offrom about 4 hours to about 24 hours.
 25. The method of claim 1, whereinthe polysaccharide-protein conjugate is white in color.